Neferine inhibits BMECs pyroptosis and maintains blood–brain barrier integrity in ischemic stroke by triggering a cascade reaction of PGC-1α

Blood–brain barrier disruption is a critical pathological event in the progression of ischemic stroke (IS). Most studies regarding the therapeutic potential of neferine (Nef) on IS have focused on neuroprotective effect. However, whether Nef attenuates BBB disruption during IS is unclear. We here used mice underwent transient middle cerebral artery occlusion (tMCAO) in vivo and bEnd.3 cells exposed to oxygen–glucose deprivation/reoxygenation (OGD/R) injury in vitro to simulate cerebral ischemia. We showed that Nef reduced neurobehavioral dysfunction and protected brain microvascular endothelial cells and BBB integrity. Molecular docking, short interfering (Si) RNA and plasmid transfection results showed us that PGC-1α was the most binding affinity of biological activity protein for Nef. And verification experiments were showed that Nef upregulated PGC-1α expression to reduce mitochondrial oxidative stress and promote TJ proteins expression, further improves the integrity of BBB in mice. Intriguingly, our study showed that neferine is a natural PGC-1α activator and illustrated the mechanism of specific binding site. Furthermore, we have demonstrated Nef reduced mitochondria oxidative damage and ameliorates endothelial inflammation by inhibiting pyroptosis to improve BBB permeability through triggering a cascade reaction of PGC-1α via regulation of PGC-1α/NLRP3/GSDMD signaling pathway to maintain the integrity of BBB in ischemia/reperfusion injury.

To verify the protective effect of Nef on BMECs: we exposed the bEnd.3 cells (BMECs of mouse) to OGD/R injury to mimic BMECs damage during cerebral I/R in vitro.Cells viability was evaluated using CCK-8 assay.As time progressed: cell viability declined (Fig. 1a).In contrast: Nef treatment increased cell viability (0.1: 0.5: and 1 μM) (Fig. 1b).Nef (0.1 μM) has been already markedly reduced the LDH leakage under OGD/R condition: and the effect of reduction is more obvious with increase of the dose (Fig. 1c).The staining images were used to revealed the PI-positive cells (dead cells) (Fig. 1e).Dead cells were evidently higher in OGD/R-exposed groups and Nef reduced the death of the OGD/R-treated bEnd.3 cells (Fig. 1d,e).These data demonstrated that Nef rescued OGD-induced cell death in bEnd.3 cells.

Nef alleviated brain ischemia-reperfusion induced brain injury and improves the integrity of BBB in mice
To investigate whether Nef exhibits BBB protective effects: tMCAO mouse as an in vivo model was established (Fig. 2a).Vehicle or Nef (30 mg/kg or 60 mg/kg) were administered to mice at 2 h and 12 h after restored the blood flow.TTC staining was employed to determine the brain infarction (Fig. 2b).The infarct volume: cerebral edema volume: and cerebral water content were increased in tMCAO mice: and Nef treatment exhibited a concentration-dependent decrease (Fig. 2c-e).Neurological status of mice was assessed using the Zea Longa neurological scoring system: Nef decreased the higher scores in tMCAO mice (Fig. 2f).
According to the results of H&E: tMCAO induced brain infarction: the peripheral neuron was nuclear pyknosis accompanied by the deeper staining: the penumbra area was swollen: neuropil vacuolation: glial cell hyperplasia: and Nef administration alleviated all the adverse phenomena elicited by tMCAO (Fig. 3a): dose-dependently.We also observed cerebral vessel damage in tMCAO mice (Fig. 3b): vessel walls integrity were impaired and the edges of the vessel wall were unclear.The mice administer Nef could effectively reverse the vessel injury.Brain EB content was quantified to further assess the integrity of BBB: tMCAO mice exhibited a marked increase in EB leakage: which was effectively attenuated by administration of Nef (Fig. 3c).
Immunohistochemistry demonstrated a decline in ZO-1 and Occludin expression levels post tMCAO (Fig. 4a-c).The gene and protein expression of ZO-1 and Occludin in bEnd.3 cells exposed to OGD/R.Consistently: Nef treatment reversed the diminished gene and protein expression of ZO-1 and Occludin in bEnd.3 cells (Fig. 5a-e)).The total pipe tube branch length of tube-like structures in bEnd.3 cells was measured to examine the tube formation ability.The morphology of the cells exhibited notable differences on tube-like structure and analysis data indicated the tubular structures decline in the total length after OGD/R.However: the tube branch length in cells which treated with Nef were dose-dependently increased (Fig. 5f,g).The protective effect of Nef on BBB was further assessed by TEER measurement (Fig. 5h) and EBA assay (Fig. 5i).Endothelial barrier permeability was evaluated after exposed to 4 h of OGD.Nef obviously ameliorated the reduction in TEER values at the period of reoxygenation onset and 12 h of reoxygenation compared with OGD group (Fig. 5j).A progressive increase in EBA extravasation is evident after OGD/R: and Nef treatment reversed this effect (Fig. 5k).These data confirm that Nef not only alleviated I/R induced brain injury but also protect cerebral vessel to improves BBB integrity after ischemic stroke.

Nef activated PGC-1α to reduce mitochondrial oxidative stress and promote TJ proteins expression
Molecular docking method was used to find the target protein interacting with Nef.To gain a comprehensive understanding with the docking residue sites of Nef and PGC-1α protein: Ligplots + and PyMOL softwares were utilized to generate visual representations of the corresponding protein residue in a 2D and 3D binding site (Fig. 6a,b).The conformation with the lowest energy was selected as the optimal conformation by the dock binding free energy.The docking simulation shows that Nef forms a hydrogen bonded with GLU-291: the best negative free energy of binding (− 9.1 kcal/mol) suggesting a possible interaction between Nef and PGC-1α (Table 1).In order to corroborate the findings of molecular docking: transient transfection of bEnd.3 cells with PGC-1α siRNA was conducted: followed by evaluation of the expression of PGC-1α: NLRP3: Occludin: and ZO-1 proteins (Fig. 6c).The results indicated that the inhibitory effect of Nef on NLRP3 expression and promotion of TJ proteins expression were counteracted by genetic PGC-1α knockdown (Fig. 6d-g)).
PGC-1α is widely acknowledged as a predominant regulator of mitochondrial biogenesis and function: we evaluated mitochondrial function in bEnd.3 cells.Following OGD/R stimulation: MMP levels decreased and treated with Nef effectively reversed (Fig. 7a,b).We further assess the impact of Nef on mtROS: SOD and MDA in bend.3 cells.Notably: Nef treatment dose-dependently inhibited mtROS (Fig. 8a,b): promoted SOD (Fig. 8c) and MDA levels (Fig. 8d).These findings provide additional evidence supporting the role of Nef in reducing mtROS: improving oxidative stress damage: and enhancing mitochondrial function.Immunohistochemistry was employed to analyze the expression of PGC-1α and NLRP3 in tMCAO mice.The results show that PGC-1α expression decreased and NLRP3 expression increased in I/R mice cerebral infarction region (Fig. 9a) and Nef dramatically reversed PGC-1α and NLRP3 expression (Fig. 9b,c).
To further verified the specific signaling pathway of Nef maintain the integrity of BBB: we also detected pyroptosis related proteins expression in bEnd.3 cells after OGD/R.The proteins expression of ASC: AIM2: Caspase-1: cleaved caspase-1: NLRP3: IL-1β: IL-18 and GSDMD were increased in bEnd.3 cells exposed to OGD/R and were reversed after the treated with Nef in vitro (Fig. 10).Consistent with the in vivo results: PGC-1α expression decreased in bEnd.3 cells under OGD/R and the expression was obviously upregulated by Nef.And the inhibiting effect of Nef on NLRP3 expression were cancelled by genetic PGC-1α knockdown (Fig. 6c).Together: these results demonstrated that Nef maintain the integrity of BBB by regulating PGC1-α/NLRP3/GSDMD mediated pyroptotic pathway.

Discussion
Previous researches have been shown that Nef had a powerful anti-inflammatory and neuroprotective effects [15][16][17] : so we tried to figure out the protective evidence of Nef on blood-brain barrier in cerebral I/R instead of neuroprotection.Our study using multiple methods to indicates that Nef repairs disruptive BBB to alleviated brain injury in cerebral I/R.Further investigation provided the first evidence that Nef target activate PGC-1α: and it promotes Occludin and ZO-1 expression: reduces NLRP3 inflammasome activation and mitochondrial oxidative stress induced pyroptosis to alleviates I/R injury of bEnd.3 cells: as illustrated in Fig. 11.Study also revealed that tight junction (TJ) protein mitigates attenuation and NLRP3 increased in bEnd.3 cells when PGC-1α was silenced.Taken all together: our work lends strong evidence supporting the protective effect of Nef on brain BMECs injury and maintain blood-brain barrier integrity after cerebral I/R.
Nef exhibits a beneficial effect on BMECs growth manifested in improving cell viability and tubular structure length and reducing the LDH release.EBA leakage and TEER values in vitro: cerebral vessel damage and EB leakage in vivo were observed to evaluate the effect of Nef on integrity of the BBB.The results both provided strong evidences of its positive regulation of BBB permeability.TJ proteins represented by Occludin and ZO-1 which are the mainly constituent and essential components in maintaining the fundamental function of the BBB.Our research shows that Nef increased TJ proteins expression which down regulated by I/R injury.This is consistent with the protective effect of Nef in tMCAO mice cerebral injury: it is manifested with the cerebral infarct volume mitigated: brain edema minimized: and neurobehavioral function enhanced.) through hydrogen bonding: and the data suggested us PGC-1α may serve as the target protein mediating the protective effects of Nef on brain vascular endothelium after I/R.PGC-1α is highly expressed in BMECs 18 .In IS: PGC-1α was down-regulated: further reduced the capacity for mitochondrial oxidative phosphorylation and increased ROS production 4,5 .PGC-1α activation could promotes new angiogenesis: facilitating the delivery of oxygen and nutrients to ischemic tissues and preventing ROS overload 3 .In our research: mtROS production in bEnd.3 cells was increased and MMP values was decreased after OGD/R: all effects can be reversed by Nef.We also observed Nef could improve SOD expression and reduce MDA levels.Therefore: we infer that Nef may activate PGC-1α to inhibit the excessive aggregation of mtROS and maintain mitochondrial function.
Endogenous ROS levels may affect protein synthesis by regulating protein oxidative modification levels: so the increase of ROS results in the decrease of protein synthesis efficiency 19 .ROS promotes damage to Tight junction 20 .The upregulated of TJ proteins expression induced by Nef treatment may related to abolish the ROS overload.
ROS trigger the assembly of inflammasomes: leading to enhanced production and secretion of pro-inflammatory cytokines like IL-1β: exacerbating inflammatory response 20,21 .The activation of NLRP3 has been widely studied, it found to promote inflammatory cascade reactions: increase inflammatory factors release and aggravate vascular inflammation in IS 22,23 .The results also showed us that Nef suppressed NLRP3 inflammasome activation through activating PGC-1α and alleviated IS induced cerebral vascular endothelial injury.Up to now: it's well known that GSDMD-N as a downstream molecule in pyroptosis: genetic PGC-1α knockdown to confirmed Nef interaction target and inhibit pyroptosis to protect blood-brain barrier.It is confirmed that Nef inhibited the expression of NLRP3 inflammasome and GSDMD in IS by targeting and activating of PGC-1α to maintain the integrity of BBB.
Based on the in vivo and in vitro research findings: we have concluded that Nef exerts a BBB-protective effect by rescue the impaired cerebral microvascular endothelial cells.The protective effect seems to be mediated by activating PGC-1α to reduce the excessive aggregation of mtROS and then inhibit the activation of NLRP3 inflammasome.Notably: the beneficial effects of Nef on cerebral microvascular endothelial against brain ischemia injury was first elucidated in our study: and first revealed that promoting PGC-1α expression could inhibit BMECs pyroptosis via PGC-1α/NLRP3/GSDMD pathway.In addition: we are concerned that PGC-1α has multiple functionalities beyond its role in reducing oxidative stress.Therefore: Further investigations are warranted to elucidate the impact of Nef on mitochondrial.To address these limitations and provide a stronger theoretical foundation for the application of Nef: we plan to conduct additional experiments and investigations.

Ethics statement
This study utilized male C57BL/6 J mice procured from Changsheng Biotechnology Technology Co.: Ltd. (Liaoning: China).Standard environmental conditions were provided for the mice to minimize environmental variability.All methods were performed in accordance with the relevant guidelines and regulations: and approved by the Ethics Committee of Hubei University of Chinese Medicine (Approval No. HUCMS22702282).

Establishment of the transient middle cerebral artery occlusion (tMCAO) model
A total of 90 male C57BL/6 J mice (aged 6-8 weeks and weighing 20-22 g) were used in short-term tMCAO experiments.Six groups were formed at random: control group: sham group: vehicle group: 30 mg/kg Nef group: 60 mg/kg Nef group: and 60 mg/kg NBP group.Dl-3n-butylphthalide (NBP): a synthetic medication derived from Apium graveolens Linn seeds (celery): has been approved by the Food and Drug Administration (FDA) for phase II clinical trials in patients with acute ischemic stroke since 2016 24 .NBP has been found to possess diverse pharmacological actions: such as promoting cerebral microcirculation and improving mitochondrial function [25][26][27] .Due to its protective effect on BBB during IS 28 : it was adopted as a positive drug.Each group consisted of 12 animals that underwent a successful operation for subsequent experiments.A total of 1% of mice failed to achieve successful reperfusion following the tMCAO procedure: and approximately 20% died within one day.
The tMCAO procedure was carried out in accordance with the methods described previously 29 .To mitigate the influence of hormonal disturbances in female mice post-tMCAO surgery: only male mice were studied.Briefly: mice were anesthetized by intraperitoneal injection of pentobarbital sodium at a dose of 70 mg/kg: and a monofilament nylon suture with a round tip was inserted into the internal carotid artery via the right common carotid artery: avoid pterygopalatine artery and finally accessed the middle cerebral artery.It was placed for 45 min until the blood flow was restored by withdrawing the filament for reperfusion.The wounds were carefully sutured to prevent infection.The sham group underwent all surgical procedures: excluding the insertion of nylon sutures.Additionally: all mice were allowed free access to food and water.

Measurement of assessment
At the end of 24 h reperfusion: neurological status of mice was assessed using the Zea Longa neurological scoring system: as mentioned earlier 30 .For this study: mice with scores ranging from 1 to 3 were included as the tMCAO model mice.Quantification of the infarct volume: cerebral edema volume: and cerebral water content.At 24 h post-reperfusion: mice were euthanized by cervical dislocation.All brains were collected and divided into five sections: then immersed immediately in a 0.5% solution of 2,3,5-triphenyltetrazolium chloride (TTC: G1017: Servisebio: Wuhan: China) at 37 ℃ for approximately 30 min.A red stain appeared on normal brain tissues: while infarcted parts displayed in white.To correct the brain swelling volume due to cerebral edema: the area of the ipsilateral uninfarcted brain slice was subtracted from the contralateral hemisphere brain slice area Fluorescence images of the specimen were acquired using the Olympus fluorescence microscope FV3000 and Image J software was employed for the measurement of fluorescence intensity.The working dilution of specific antibodies can be found in Supplementary materials.

Oxygen-glucose deprivation/reoxygenation (OGD/R) establishment and drug treatment
Cells were subjected to OGD/R to simulate I/R injury.Upon reaching 80-90% confluence: bEnd.3 cells were placed in glucose-free DMEM (Procell: Wuhan: China) that had been degassed by ultrasonication.Then: cells were treated with Nef at concentrations of 0.1 μM: 0.5 μM or 1 μM: and transferred to a chamber consisting of 5% CO2 and 95% N2 for 9 h.Following the hypoxia exposure: replace the media with fresh growth medium and incubated another 12 h under normoxic conditions for reperfusion.

Cell viability assays
A colorimetric CCK-8(GK10001: GLPBIO: CA: USA) assay was utilized for cell viability measurement.After OGD/R treatment: cell viability data exserts a relative percentage change compared to the untreated control group.Furthermore: a commercial assay kit (LDH: C0016: Beyotime: Shanghai: China) was employed to measure LDH release: a marker of cellular toxicity.Briefly: 50 µl of culture medium from each group was transferred to another 96-well plate: followed by LDH measurement using the manufacturer-provided test reagent.Additionally: the visualization of live and dead cells was conducted employing a Live/Dead assay kit (BB-4126: BestBio: Shanghai: China).Fluorescence images of the specimen were acquired using the Olympus fluorescence microscope FV3000 and Image J software was employed for the measurement of fluorescence intensity.

Tube formation assay
The analysis of tube formation was conducted using Matrigel basement membrane matrix with growth factorreduced (082,701: ABWbio: Shanghai: China).Briefly: a volume of 60 μL of Matrigel matrix was added to a 24-well plate.The pretreated bEnd.3 cells were then seeded and incubated for 6 h.Microscope photos were then obtained from three randomly chosen optical fields at a magnification of × 100.Analysis of the tube branch length was conducted by an uninformed experimenter: utilizing the "Angiogenesis Analyzer" plugin in Image J software.Each experiment was conducted a minimum of three times.

Endothelial barrier permeability measurement
To assess endothelial barrier permeability: transmembrane electrical resistance (TEER) values of endothelial cell monolayer were measured using transmembrane resistance measuring instruments (EVOM2: WPI: Florida: USA) at time points of OGD: reoxygenation initiation: and 12 h of reperfusion.TEER values were calculated as followed: TEER (Ω × cm 2 ) = (total resistance − blank resistance) (Ω) × insert area (cm 2 ).The flux of Evans blue-labeled albumin (EBA: 1% BSA + 167.5 µg/ml Evans blue; 67 kDa) across the cell monolayer was measured according to previous report 33 .Following 12 h reoxygenation: 50 μL of EBA was added to the transwell inserts.Over the next six hours: media from the lower chambers from each group were collected hourly to measure optical density at 630 nm.

Oxidative stress assay
Levels of superoxide dismutase (SOD: S0101S: Beyotime: Shanghai: China) and malondialdehyde (MDA: S0131S: Beyotime: Shanghai: China) were measured to assess intracellular oxidative stress using commercially available kits.The experiments were conducted in triplicate using 6-well plates for each condition.

Western blot analysis
Cellular protein extraction was accomplished using Cell lysis buffer for Western and IP (P0013: Beyotime: Shanghai: China) in accordance with the manufacturer's instructions.SDS-PAGE was employed to separate the protein samples (30 μg per group): followed by transferred onto a PVDF membrane (0.45 μm pore size: Millipore: MA: USA).The membrane blocked for 30 min and incubated overnight with the primary antibodies listed in Table 2 at 4 ℃.After that: the membrane were interacted with the respective secondary antibody for 1.5 h.β-actin was used as a loading reference.The protein bands were detected using ECL method (MA0186-1: Meilunbio: Dalian: China) and developed with gel densitometric scanning.

Quantitative real-time polymerase chain reaction (qRT-PCR)
TRIzol reagent (BS258A: Biosharp: Anhui: China) was employed to obtain total RNA from bEnd.3 cells.Subsequently: DNA contamination was eliminated from the total RNA: and cDNA was synthesized using the ABScript II cDNA First Strand Synthesis Kit (RK20400: ABclone: Wuhan: China).In the end: cDNA was mixed with specific primers and qRT-PCR was performed using TOYOBO SYBR Green Realtime PCR Master Mix (QPK-201: TOYOBO: Japan) to validate the expression of specific mRNAs.The specific primers listed in Table 3 were utilized for PCR amplification of the cDNA fragment.The 2 − ΔΔCt method was used to calculate the relative gene expression levels: and β-actin expression served as endogenous internal control.

Mitochondrial ROS (mtROS) measurement
The mtROS level was assessed with MitoSOX Red Mitochondrial Superoxide Indicator (M36008: Invitrogen: CA: USA).Following OGD/R injury: the cells underwent centrifugation at 1000 g for 5 min and were then washed with PBS.Mito-Tracker Red CMXRos and Hoechst 33,342 were then added and incubated in dark for 30 min.Fluorescence images of the specimen were acquired using the Olympus fluorescence microscope FV3000 and Image J software was employed for the measurement of fluorescence intensity.

Mitochondrial membrane potential (MMP) measurement
Mitochondrial Membrane Potential Assay Kit (E-CK-A301: Elabscience: Wuhan: China) was employed to assess the MMP in bEnd.3 cell.Briefly: JC-1 dye solution was added to cells and incubated for 20 min after OGD/R injury: followed by 5 min incubation with Hoechst 33,342.Olympus Fluorescence FV3000 microscope was employed to observe the fluorescence and Image J software was employed for the measurement of fluorescence intensity.

Molecular docking
To initiate the molecular docking process: the PGC-1α protein structure (PDBID: 3B1M) was imported from the RSCB PDB database in PDB format: while the 3D structure of Neferine was obtained from PubChem.Using PyMOL software: water molecules were removed: and the ligand was isolated.After employing AutoDock Tools 1.5.6 to perform tasks such as hydrogen addition: charge calculation: and atomic type assignment.Subsequently: AutoDock Vina 1.1.2was utilized for molecular docking simulations to investigate the binding characteristics between the Nef ligand and PGC-1α protein.The binding energy: which serves as an evaluation index for molecular docking: was calculated.The optimal conformations of Nef and PGC-1α were determined by analyzing the clusters within the docking results: which were selected based on their respective binding energies.Typically: a lower docking energy indicates a stronger interaction force between the components: and a threshold of -7 kcal/ mol is often used.The epitope with the lowest affinity scores predicted by AutoDock Vina was subjected to Lig-Plot + and PyMol for further visualization of the interactions.

Short interfering (Si) RNA and plasmid transfection
Cells cultivated in a 6-well plate with OPTI medium (31,985,062: Invitrogen: CA: USA) were transfected with either PGC-1α siRNA (3′-CCG CAA UUC UCC CUU GUA UTT-5′) or negative control siRNA (3′-ACG UGA CAC GUU CGG AGA A-5′) using Lipofectamine 3000 transfection reagent (L3000001: Invitrogen: CA: USA).The siRNAs were obtained from Sangon (Shanghai: China).Following 12 h incubation: all medium was substituted with growth medium: and cells were cultured for an additional 24 h.Cells were then subjected to OGD/R injury and harvested for Western blot analysis.

Table 1 .
Dock binding free energies (△Gb) and bonds of the docked compounds against proteins.

Table 2 .
The protein antibodies.

Table 3 .
The primer pairs.